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Clinical Chemistry 45: 1391-1396, 1999;
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(Clinical Chemistry. 1999;45:1391-1396.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Development of a Chemiluminescence Competitive PCR for the Detection and Quantification of Parvovirus B19 DNA Using a Microplate Luminometer

Fabiana Fini1, Giorgio Gallinella2, Stefano Girotti1, Marialuisa Zerbini2 and Monica Musiani2,a

1 Institute of Chemical Science and
2 Department of Clinical and Experimental Medicine, Division of Microbiology, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy.
a Author for correspondence. Fax 0039-051-341632; e-mail musiani{at}med.unibo.it

Background: Quantitative PCR of viral nucleic acids can be useful clinically in diagnosis, risk assessment, and monitoring of antiviral therapy. We wished to develop a chemiluminescence competitive PCR (cPCR) for parvovirus B19.

Methods: Parvovirus DNA target sequences and competitor sequences were coamplified and directly labeled. Amplified products were then separately hybridized by specific biotin-labeled probes, captured onto streptavidin-coated ELISA microplates, and detected immunoenzymatically using chemiluminescent substrates of peroxidase. Chemiluminescent signals were quantitatively analyzed by a microplate luminometer and were correlated to the amounts of amplified products.

Results: Luminol-based systems displayed constant emission but had a higher detection limit (100–1000 genome copies) than the acridan-based system (20 genome copies). The detection limit of chemiluminescent substrates was lower (20 genome copies) than colorimetric substrates (50 genome copies). In chemiluminescence cPCR, the titration curves showed linear correlation above 100 target genome copies. Chemiluminescence cPCR was positive in six serum samples from patients with parvovirus infections and negative in six control sera.

Conclusions: The chemiluminescence cPCR appears to be a sensitive and specific method for the quantitative detection of viral DNAs.




The following articles in journals at HighWire Press have cited this article:


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 J. Mol. Diagn.Home page
S. Schorling, G. Schalasta, G. Enders, and M. Zauke
Quantification of Parvovirus B19 DNA Using COBAS AmpliPrep Automated Sample Preparation and LightCycler Real-Time PCR
J. Mol. Diagn., February 1, 2004; 6(1): 37 - 41.
[Abstract] [Full Text]

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 J. Clin. Microbiol.Home page
T. C. Harder, M. Hufnagel, K. Zahn, K. Beutel, H.-J. Schmitt, U. Ullmann, and P. Rautenberg
New LightCycler PCR for Rapid and Sensitive Quantification of Parvovirus B19 DNA Guides Therapeutic Decision-Making in Relapsing Infections
J. Clin. Microbiol., December 1, 2001; 39(12): 4413 - 4419.
[Abstract] [Full Text] [PDF]


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